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Am J Physiol Gastrointest Liver Physiol 291: G1180-G1186, 2006. First published July 27, 2006; doi:10.1152/ajpgi.00224.2006
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NEUROREGULATION AND MOTILITY

Serotonergic modulation of murine fundic tone

Lin Xue,1 Michael Camilleri,1 G. Richard Locke, III,1 Jan A. J. Schuurkes,2 Ann Meulemans,2 Bernard J. Coulie,2 Joseph H. Szurszewski,1 and Gianrico Farrugia1

1Enteric NeuroScience Program, Mayo Clinic College of Medicine, Rochester, Minnesota; and 2Johnson & Johnson Pharmaceutical Research and Development, Beerse, Belgium

Submitted 19 May 2006 ; accepted in final form 26 July 2006


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Fundic tone is maintained through a balance of excitatory and inhibitory input to fundic smooth muscle. The aim of this study was to determine the role of serotonin (5-HT) and 5-HT receptors in modulating murine fundic tone. Muscle strips were prepared from the murine fundus. Intracellular recordings were made from circular smooth muscle cells, and the effects of 5-HT on tone and excitatory and inhibitory junction potentials evoked by electrical field stimulation (EFS) were determined. 5-HT induced a concentration-dependent contraction and smooth muscle depolarization that was tetrodotoxin resistant. The 5-HT1B/D receptor antagonists GR-127935 and BRL-155172 significantly inhibited 5-HT-induced contractions. The 5-HT1B/D agonist sumatriptan contracted murine fundic muscle. The 5-HT1A receptor agonist buspirone relaxed fundic smooth muscle, and the relaxation was inhibited by WAY-100135 but not by N{omega}-nitro-L-arginine or tetrodotoxin. 5-HT enhanced both the excitatory and inhibitory responses to EFS. The 5-HT3 receptor antagonist MDL-72222 partly inhibited both the excitatory and inhibitory response elicited by EFS, whereas the 5-HT4 receptor antagonist GR-113808 partly inhibited the EFS-evoked inhibitory response. The 5-HT reuptake inhibitor fluoxetine contracted smooth muscle strips, a contraction that was partially inhibited by GR-127935 and abolished by tetrodotoxin. In conclusion, the data suggest that 5-HT modulates murine fundic contractile activity through several different receptor subtypes. Sustained release of 5-HT maintains fundic tone through postjunctional 5-HT1B/D receptors. 5-HT3 receptors modulate excitatory neural input to murine fundic smooth muscle, and both 5-HT3 and 5-HT4 receptors modulate inhibitory neural input to murine fundic smooth muscle.

receptors; smooth muscle; enteric nerves; fundic accommodation; serotonin

THE STOMACH WALL relaxes to accommodate the entry of content. Relaxation allows the accommodation of a large volume of content without a marked increase in intragastric pressure (7, 15). Although there is evidence that the antrum accommodates after a meal, gastric accommodation occurs predominantly in the proximal stomach, especially in the fundus. The fundic integrated relaxatory response to a meal is known as fundic accommodation. The accommodation response is mediated through an extrinsic vagovagal reflex pathway and a nonvagal intrinsic reflex pathway. Both pathways act on intrinsic noncholinergic, nonadrenergic (NANC) neurons in the fundus and other parts of the stomach wall to relax smooth muscle cells (1, 3, 26, 28). In the fasted state, well-established mediators of gastric fundic tone are vagally mediated cholinergic input (2, 29) and NANC inhibitory input (11, 12). The regulation of fundic tone is also mediated through serotonin (5-HT). In humans, sumatriptan, a 5-HT1B/D agonist, relaxes the fundus (24, 25). In mice, in vivo, buspirone relaxed the fundus, whereas sumatriptan contracted it (29). There are two sources for 5-HT in the gut: release from enterochromaffin (EC) cells in the mucosa and from myenteric interneurons (21). Additionally, in some species, including mice, mast cells contain 5-HT (4). It is well established that endogenous 5-HT released from mucosal EC cells activates intrinsic primary afferent neurons to initiate the peristaltic reflex in the small intestine (6, 14). A similar pathway is proposed in the stomach: food stimulates mucosal EC cells, which in turn activate primary afferent neurons initiating gastric accommodation via the vagovagal reflex pathway. In contrast, the role of 5-HT in mediating the nonvagally mediated component of fundic accomodation and the potential 5-HT receptors involved are less well understood. Several different 5-HT receptors are expressed in the gastrointestinal tract, and each receptor is coupled to a different signaling cascade (13). It is therefore likely that 5-HT will have different effects on fundic smooth muscle tone according to the particular receptor and the related signaling cascade activated. The aims of the present study were to determine the role of 5-HT and the specific 5-HT receptors involved in regulating enteric nerve and smooth muscle control of murine fundic tone. The data show that 5-HT modulates murine fundic tone through activation of multiple 5-HT receptor subtypes including non-neuronal 5-HT1A and 5-HT1B/D receptors and neuronal 5-HT3 and 5-HT4 receptors.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Animals. Adult male 129 SvEv mice (Tocanic, Gemantown, NY) of 6–8 wk old were euthanized with CO2 gas with the prior approval of the Mayo Animal Care and Use Committee. Mice were fasted overnight with free access to water. After the abdomen was opened, the stomach was removed and placed in preoxygenated Krebs solution. The stomach was opened along the lesser curvature, and all contents were removed. The fundus was defined as the proximal portion of the stomach, which has a different appearance (whiter) than the rest of the stomach. The fundus was removed from the rest of the stomach and transferred to fresh, oxygenated Krebs solution. The mucosa was carefully removed under direct vision using a binocular microscope, and full-thickness muscle strips (0.5–0.8 x 0.2 mm) were prepared with the long axis cut parallel to the circular muscle layer.

Electrophysiology. Muscle strips were placed in a recording chamber with the circular muscle facing upward. One end of the muscle strip was pinned to the Sylgard (Dow Corning, Midland, MI)-coated floor of the chamber to record intracellular electrical activity, whereas the other end was attached to an isometric force transducer to record mechanical activity. The chamber (3-ml volume) was perfused with prewarmed (37°C) and preoxygenated Krebs solution at a constant flow rate of 3 ml/min. After an equilibration period of at least 2 h, muscle strips were stretched to an initial tension of 100 mg above the baseline tension. Recordings of intracellular electrical activity from smooth muscle cells were obtained using glass capillary microelectrodes filled with 3 M KCl and with resistances ranging from 50 to 80 M{Omega}. Intracellularly recorded potentials were amplified using a WPI M-707 amplifier (WPI, New Haven, CT) and displayed on an oscilloscope (Tektronix 5113, Tektronix, Beaverton, OR). Force was measured isometrically and amplified with a bridge circuit amplifier. Both electrical and mechanical signals were displayed and recorded on a Windows-based computer through a Digidata 1322A (Axon Instruments, Foster City, CA) using Axoscope 8.1 software (Axon Instruments). Two platinum wires were placed parallel to the long axis of the preparation and connected to a square-wave stimulator (Grass 588, Grass Instruments, Quincy, MA) and a stimulus isolation unit (Grass SIU 5A) to apply external field stimulation (EFS). Individual electrical pulses were of 0.35-ms duration and 100- to 150-V intensity. The ranges of frequency used were 1–30 Hz.

Solutions and drugs. Krebs solution had the following ionic composition (in mM): 127.4 Na+, 5.9 K+, 2.5 Ca2+, 1.2 Mg2+, 134 Cl–, 15.5 HCO3–, 1.2 H2PO4–, and 11.5 glucose. The solution was aerated with 97% oxygen-3% CO2 and maintained at pH 7.4. 5-HT, atropine sulphate, acetylcholine chloride, N{omega}-nitro-L-arginine (L-NNA), and tetrodotoxin were obtained from Sigma (St. Louis, MO). Buspirone hydrochloride, GR-127935 hydrochloride, BRL-15572 hydrochloride, (S)-WAY-100135 dihydrochloride, methiothepin maleate, MDL-72222, GR-113808, and SB-269970 hydrochloride were obtained from Tocris (Ellisville, MO). Sumatriptan succinate (Imitrex) was obtained from GlaxoWellcome (Research Triangle Park, NC). Doses of 5-HT were separated by at least a 30-min interval. With this dose interval, no desensitization to the response to 5-HT was observed.

Statistical analysis. All observed values are expressed as means ± SE. The numbers of cells recorded from muscle strip preparations are designated by n in the RESULTS unless expressly stated otherwise as numbers of preparations. The percent relaxation was compared with the initial active tension. Statistical significance was determined using Student's t-test. A P value of <0.05 was considered as significant.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Effect of exogenous 5-HT. The resting membrane potential of fundic circular smooth muscle cells was –47.4 ± 0.5 mV (n = 78 preparations). No spontaneous phasic contractile activity was recorded from fundic circular smooth muscle. Perfusion of 5-HT induced a concentration-dependent smooth muscle membrane potential depolarization (1 µM 5-HT, 4.5 ± 0.6 mV, n = 12, P < 0.05; Fig. 1, A, a, top trace, and B,a). The depolarization was accompanied by a concentration-dependent transient tonic contraction (Fig. 1A,a, bottom trace). The contraction was first seen at 1 nM and increased in amplitude with increasing concentrations of 5-HT (Fig. 1B,b; n = 5 at each concentration, P < 0.05 at 10–8 M and higher). Pretreatment of the muscle strips with atropine (1 µM, n = 10) or tetrodotoxin (100 nM, n = 5; Fig. 2) did not inhibit the 5-HT-induced depolarization or contraction, suggesting that the 5-HT-induced response was not neuronally mediated. The 5-HT-induced depolarization gradually returned to baseline within 5 min, but the contraction persisted for a longer period (Fig. 1A; average time: 27 ± 1.4 min, n = 10).


Figure 1
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  		Fig. 1. Effect of serotonin (5-HT) and the 5-HT1B/D receptor antagonist GR-127935 on murine fundic smooth muscle electrical and mechanical activity. Application of 5-HT (1 µM) to a fundic smooth muscle strip while circular muscle electrical and mechanical activity were recorded induced a depolarization and relaxation (A,a, top and bottom traces, respectively). The 5-HT1B/D receptor antagonist GR-127935 (10 µM) inhibited the depolarization and partly inhibited the contraction induced by 5-HT (A,b). The effect of 5-HT on fundic smooth muscle electrical and mechanical activity was concentration dependant. Exogenous application of 5-HT (1 nM–10 µM) evoked a concentration-dependent depolarization (B,a) and an associated contractile response (B,b). The values of depolarization data were obtained from 15–30 cells/concentration, and the values of contraction data were obtained from 5–10 preparations/concentration. Data for contraction are normalized to the response to 1 µM 5-HT. *P < 0.05.

 

Figure 2
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  		Fig. 2. Effect of the 5-HT receptor antagonists atropine and tetrodotoxin (TTX) on the increase in tone induced by 5-HT. The 5-HT1B/D receptor antagonist GR-127935 (10 µM) and the 5-HT1D antagonist BRL-15572 (3 µM) partly inhibited the contractile response to 5-HT (1 µM), whereas the 5-HT2 antagonist methiothepin (10 µM), the selective 5-HT7 antagonist SB-269970 (10 nM), atropine (1 µM), and TTX (300 nM) had no effect on the 5-HT-induced contraction, suggesting a postjunctional location of 5-HT1B/D receptors. Values are means ± SE; n = 5–12 preparations/drug. *P < 0.05.

 
A 5-HT-induced relaxation was observed in 2 of the 53 tissues tested. Of the two relaxations observed, one was induced by 10 nM 5-HT and the other was induced by 1 µM 5-HT. In both cases, the relaxation was not concentration dependent. Because of the rarity of the 5-HT induced relaxation, subsequent experiments focused on the 5-HT-induced contractile response. As can be seen in Fig. 1, 1 µM 5-HT reproducibly resulted in a robust and stable contractile response. Therefore, this concentration was used to carry out further experiments. All changes observed with other 5-HT receptor agonists and antagonists were compared with the contraction induced by 5-HT.

Effects of 5-HT receptor antagonists on 5-HT contractile responses. Antagonists selective for 5-HT receptor subtypes were used to determine the 5-HT receptors that mediated the 5-HT induced fundic smooth muscle contraction and depolarization. GR-127935 (10 µM), a 5-HT1B/D receptor antagonist, inhibited the depolarization (3.6 ± 1.2 vs. 0 ± 0 mV, n = 3, P < 0.001) and contraction evoked by 1 µM 5-HT (72 ± 8% decrease, n = 9, P < 0.05; Figs. 1A,b and 2). The 5-HT-induced contraction and depolarization (Fig. 2) and the inhibition of the 5-HT-induced contraction and depolarization by GR-127935 were tetrodotoxin insensitive. These data suggest that 5-HT1B/D receptors partly mediated the effect of 5-HT on contraction and depolarization and that the receptors were located postjunctionally. BRL-155172, a 5-HT1D receptor antagonist with a lower affinity for the 5-HT1B receptor, also significantly inhibited the 5-HT-induced increase in tone (55 ± 8% inhibition, n = 3, P < 0.05; Fig. 2).

Postjunctional 5-HT2 receptors mediate contractile events in the guinea pig ileum (5). The 5-HT2 receptor antagonist methiothepin (0.1–10 µM) was used to determine if, together with 5-HT1B/D receptors, 5-HT2 receptors also participate in the murine fundic smooth muscle contractile response to 5-HT. At a concentration of up to 10 µM, methiothepin had no effect on the contractile concentration response to 5-HT (12 ± 6.1% inhibition, n = 5, P > 0.05; Fig. 2). Another 5-HT receptor subtype reported to be expressed on smooth muscle is the 5-HT7 receptor (18). The effect of the 5-HT7 receptor antagonist SB-269970 was tested on the 5-HT-induced fundic contraction. SB-269970 (10 nM) had no effect on the 5-HT-induced fundic contraction (2 ± 9% inhibition, n = 3, P > 0.05; Fig. 2).

Effects of endogenous 5-HT on fundic tone. When the 5-HT1B/D receptor antagonist GR-127935 (100 nM–10 µM) was applied to murine fundic circular smooth muscle strips without pretreatment with 5-HT, a slow gradual decrease (over 20–25 min) in murine fundic circular smooth muscle tone was observed (21 ± 1.3% decrease, n = 6, P < 0.05; Fig. 3A). These data suggested that there was an endogenous baseline sustained release of 5-HT, likely acting on 5-HT1B/D receptors to regulate fundic tone. Because the mucosa was removed from the preparations, the source of 5-HT was likely neuronal. Fluoxetine, a 5-HT reuptake inhibitor, was used to increase the local concentration of endogenously released 5-HT. Fluoxetine (10 µM) resulted in a significant increase in tone (25.4 ± 2.8% increase, n = 5, P < 0.05; Fig. 3B,a). The increase in tone was partly, but not completely, inhibited by GR-127935 (10 µM), from 28.3 ± 3.8% to 15 ± 1.7% (n = 3, P < 0.05; Fig. 3B,b), suggesting that other receptors also were involved in the endogenous 5-HT regulation of fundic tone. To further test the hypothesis that the source of endogenous 5-HT production was neuronal, tissues were incubated with tetrodotoxin before the application of fluoxetine. In contrast to the contractile response to exogenous 5-HT, the fluoxetine-induced contraction was tetrodotoxin sensitive. Tetrodotoxin (100 nM) completely abolished the fluoxetine-induced increase in tone (0 ± 0 increase in tone, n = 3, P > 0.05) and lowered fundic tone to below basal levels (Fig. 3B,c), suggesting both the inhibition of endogenous 5-HT release as well other excitatory neurotransmitters such as acetylcholine, which has been previously shown to modulate baseline tone (29).


Figure 3
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  		Fig. 3. Effect of endogenous 5-HT on fundic tone. Application of the 5-HT1B/D receptor antagonist GR-127935 (1 µM) resulted in a gradual decrease in fundic tone (A) that was reversible when GR-127935 was washed out. Application of the selective 5-HT reuptake inhibitor fluoxetine (10 µM) induced a transit reversible contraction (B,a) that was inhibited by the 5-HT1B/D antagonist GR-127935 (B,b). TTX (100 nM) completely inhibited the fluoxetine-induced contractile response (B,c) suggesting that an endogenous sustained release of 5-HT from enteric neurons modulates murine fundic tone, at least partly through 5-HT1B/D receptors.

 
Effects of 5-HT1A and 5-HT1B/D receptors on 5-HT-induced contractions. The dominant effect of exogenous application of 5-HT to murine fundic muscle strips in the present experiments was a contractile response; however, a relaxation was seen in two preparations. A concentration-dependent relaxation has been observed in the guinea pig fundus (19), suggesting the possibility that the 5-HT-modulated relaxation in the mouse fundus was masked by a stronger contractile response to exogenous 5-HT. Buspirone and sumatriptan were used to determine whether specific 5-HT receptor subtypes could potentially modulate a local relaxatory response to 5-HT.

Sumatriptan (1 µM) contracted the fundus (37.8 ± 9.3% increase, n = 4, P < 0.05; Fig. 4A). The contraction induced by sumatriptan was inhibited by GR-127935 (10 µM) and BRL-15572 (3 µM) (Fig. 4A). Buspirone relaxed fundus smooth muscle. The maximal relaxation response induced by buspirone was 33 ± 6.7% (10 µM, n = 5, P < 0.05; Fig. 4B). The 5-HT1A receptor antagonist WAY-100135 (3 µM) inhibited the buspirone-evoked relaxation (56 ± 9% inhibition of relaxation, n = 3, P < 0.05), suggesting a 5-HT1A-modulated effect (Fig. 4B). Because nitric oxide is the predominant inhibitory neurotransmitter in the gastrointestinal tract (23), preparations were pretreated with L-NNA (100 µM), a nitric oxide synthase inhibitor, for 15 min, and buspirone was then applied to determine whether the relaxation induced by buspirone was mediated through nitric oxide. L-NNA had no effect on the buspirone-induced relaxation, suggesting that the relaxation was not mediated through nitric oxide (9 ± 3% inhibition of relaxation, n = 5, P > 0.05; Fig. 4B).


Figure 4
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  		Fig. 4. Mechanisms of action of the 5-HT1B/D receptor agonist sumatriptan (A) and the 5-HT1A receptor agonist buspirone (B). The contractile response to sumatriptan (1 µM) was partly inhibited by GR-127935 (10 µM) and BRL-15572 (3 µM). The decrease in tone induced by buspirone (1 µM) was partly inhibited by the 5-HT1A receptor antagonist WAY-100135 (3 µM) but not N{omega}-nitro-L-arginine (L-NNA; 100 µM), suggesting that the observed relaxation was not through the nitric oxide pathway. Values are means ± SE; n = 3–5 preparations/drug. *P < 0.05.

 
Neuronal effects of 5-HT. EFS elicited electrical and mechanical responses. The electrical response consisted of a biphasic response: an excitatory junction potential (EJP) and an inhibitory junction potential (IJP). The mechanical response consisted of a contraction followed by a relaxation (Fig. 5A). In the murine fundus, the contractile response is predominantly cholinergic, whereas the relaxatory response is predominantly mediated through nitric oxide released from NANC inhibitory motor neurons (27). Pretreatment of murine fundic muscle strips with 5-HT (10 nM–1 µM) significantly enhanced the amplitude of the EFS-elicited EJPs and IJPs and the accompanied mechanical response (Fig. 5B; EJP: 4.9 ± 0.8 mV in controls compared with 7.5 ± 0.4 mV with 10nM 5-HT, n = 10, P < 0.05; IJP: 7.6 ± 0.7 mV in controls compared with 13 ± 1.6 mV with 10 nM 5-HT, n = 10, P < 0.05). The enhanced electrical response to exogenous 5-HT suggested that 5-HT may partly modulate EFS-induced enteric neurotransmission in the murine fundus. To characterize the receptor subtypes involved in this response, antagonists to the major classes of 5-HT receptors expressed in the gastrointestinal tract were used, and EFS was applied to elicit enteric nerve responses. Methiothepin (a 5-HT2 receptor antagonist) and SB-26997 (a 5-HT7 receptor antagonist) had no effect on the excitatory and inhibitory responses to EFS (Fig. 6). MDL-72222 (10 µM), a 5-HT3 receptor antagonist, significantly inhibited the amplitude of both the EJP and IJP (EJP: 6.6 ± 1.3 vs. 3.1 ± 1.1 mV; IJP: 15 ± 2.5 vs. 8.4 ± 1.9 mV, n = 5, P < 0.05; Figs. 6 and 7). GR-113808 (1 µM), a 5-HT4 receptor antagonist, significantly inhibited the EFS-elicited IJP and the accompanying relaxation (IJP: 9.6 ± 0.6 vs. 1.5 ± 0.4 mV, n = 4, P < 0.05; Figs. 6 and 8). The contractile response to EFS was increased in the presence of GR-113808. However, there was no effect on the EJP (5.7 ± 1.6 vs. 6.9 ± 1.9 mV, n = 4, P > 0.05), suggesting that the increase in tone was likely due to inhibition of the relaxation (Figs. 6 and 8).


Figure 5
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  		Fig. 5. Effect of 5-HT on electrical field stimulation (EFS)-induced excitatory and inhibitory responses. EFS (30 Hz, 0.35 ms x 6) elicited both a depolarization [excitatory junction potential (EJP)] and hyperpolarization [inhibitory junction potential (IJP)] accompanied by a contraction and relaxation, respectively. The top traces in A and B show the electrical response, and the bottom traces in A and B show the mechanical response. A: response to EFS under control (CTRL) conditions; B: response recorded from the same cell after the application of 5-HT (10 nM). A higher concentration of 5-HT resulted in a stronger contraction and loss of impalement. C: overlapped traces for both electrical (a) and mechanical (b) responses to EFS before and after the administration of 5-HT showing that 5-HT increased EJP and IJP amplitude and the resulting smooth muscle mechanical response.

 

Figure 6
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  		Fig. 6. Summary of the effect of 5-HT and 5-HT receptor antagonists on the EFS-elicited EJP and IJP. 5-HT (10 nM) enhanced both the EJP and IJP, and the 5-HT3 receptor antagonist MDL-72222 (10 µM) inhibited both the EJP and IJP, whereas the5-HT4 receptor antagonist GR-113808 (10 µM) inhibited the IJP only. The 5-HT2 receptor antagonist methiothepin (10 µM) and the 5-HT7 receptor antagonist SB-269970 (10 nM) had no effect on the EJP and IJP. Each column shows data obtained from 5–10 preparations. *P < 0.05.

 

Figure 7
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  		Fig. 7. Effect of the 5-HT3 receptor antagonist MDL72222 on the EFS-elicited EJP and IJP. MDL-72222 (10 µM) inhibited both the EFS-evoked EJP and IJP. A and B were obtained from the same cell before (A) and after (B) the application of MD-L72222 (10 µM). The top traces in A and B show the electrical response to EFS, and the bottom traces in A and B show the mechanical response.

 

Figure 8
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  		Fig. 8. Effect of the 5-HT4 antagonist GR-113808 on the EFS-elicited EJP and IJP. GR-113808 (1 µM) inhibited the EFS-evoked IJP but not EJP. The contractile response was enhanced, likely due to the inhibition of the IJP. A and B were obtained from the same cell before (A) and after (B) the application of GR-113808 (1 µM). The top traces in A and B show the electrical response to EFS, and the bottom traces in A and B show the mechanical response.

 
To attempt to determine the location of 5-HT3 and 5-HT4 receptors, fundic tissue was pretreated with hexamethonium to block nicotinic neurotransmission. Hexamethonium (100 µM) did not alter the effect of either MDL-72222 or GR-113808 on EJP and IJP amplitudes. Both MDL-72222 and GR-113808 still significantly altered the EFS-evoked excitatory (10 µM MDL-72222: 37 ± 8%; 3 µM GR-113808: 132 ± 5%) and inhibitory responses (MDL-72222: 57 ± 16%; GR-113808: 66 ± 4%, n = 3 for each drug).


   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
The main finding of the present study was that 5-HT modulates murine fundic contractility and does so through multiple 5-HT receptor subtypes. At least four receptor subtypes, namely, 5-HT1B/D, 5-HT1A, 5-HT3, and 5-HT4 receptors, mediate the effect of 5-HT on murine fundic neuromuscular function. Modulation of the excitatory and inhibitory input to murine fundic smooth muscle is achieved through the balance of the specific subtypes of 5-HT receptors that are activated.

In a previous in vivo study (29) on murine fundic tone, buspirone, a 5-HT1A receptor agonist, relaxed the murine fundus, and sumatriptan, a 5-HT1B/D receptor agonist, contracted the murine fundus, suggesting a role for 5-HT and 5-HT1 receptors in the regulation of murine fundic tone. Data from the present study confirm and expand these findings. Exogenous 5-HT induced a fundic contraction that was inhibited by the 5-HT1B/D receptor antagonists GR-127935 and BRL-15572 and was tetrodotoxin insensitive. These data suggest that the location of 5-HT1B/D receptors is non-neuronal. In vitro, as in vivo, sumatriptan contracted the murine fundus, and the contraction was inhibited by GR-127935 and BRL-15572 pretreatment. The contractile effect of sumatriptan on the murine fundus seen in vivo (29) and in this study is in contrast to the relaxatory effect seen in humans and cats (11, 24), suggesting a species-specific effect of sumatriptan. In muscle strip experiments, a 5-HT-induced contraction in the fundus has been previously described in several species (8–10, 17, 22). For example, in the rat fundus, 5-HT contracts the fundus through 5-HT1 receptors but not 5-HT2 receptors (8). In the canine fundus, the predominant contractile effect of 5-HT is mediated by 5-HT2A receptors (17).

In contrast to sumatriptan, buspirone relaxed the murine fundus in vivo (29) and in vitro in this study. Buspirone relaxed murine fundic smooth muscle strips in a concentration-dependent manner that was not inhibited by L-NNA, an inhibitor of nitric oxide synthase, or by tetrodotoxin, suggesting a non-neuronal serotonergic non-nitric oxide-mediated mechanism to relax the murine fundus through 5-HT1A receptors. Relaxation of the fundus by 5-HT1 receptors has been previously reported in the guinea pig. 5-HT induced fundic relaxation under resting muscle tension, a relaxation that was also proposed to be mediated by 5-HT1 subtype receptors and was also tetrodotoxin insensitive, suggesting a direct effect on smooth muscle (19, 20). The available data suggest that 5-HT has multiple non-neuronally mediated effects on fundic tone with the balance between contractile and a relaxatory responses to 5-HT determined by the differential expression of 5-HT receptor subtypes in different species. For example, in the guinea pig, 5-HT1A receptors may be more highly expressed, resulting in a predominant relaxatory response to 5-HT. In the murine fundus, a 5-HT1A receptor-mediated relaxation may participate in regulating muscle tone, but the stronger contractile response to 5-HT masks the 5-HT1A receptor-induced relaxation. It is still, however, unclear why, in the present study, WAY-100135 (a 5-HT1A receptor antagonist) did not increase the excitatory response, as would be expected if an underlying relaxatory response to 5-HT was blocked. It is possible that either the relaxatory response is only a minor component of the overall response to 5-HT or that buspirone is not acting solely through 5-HT1A receptors.

In addition to the direct effects of 5-HT on smooth muscle, 5-HT also appears to modulate murine fundic tone by altering neuronal activity. The 5-HT1B/D receptor antagonist GR-127935 consistently decreased murine fundic smooth muscle strip tension when applied to the bath in the absence of exogenous neuronal stimulation. This result suggests that there is a baseline release of 5-HT that modulates fundic tone through 5-HT1B/D receptors. This hypothesis was supported by the experiments carried out using fluoxetine, a serotonin reuptake transporter inhibitor. Fluoxetine induced a reversible contraction that was partially inhibited by GR-127935, again suggesting that there is a basal release of 5-HT that is involved in the maintenance of fundic tone. The effect of fluoxetine was partially but not completely inhibited by GR-127935, suggesting that, together with 5-HT1B/D receptors, other receptors also mediate the effect of endogenous 5-HT on murine fundic tone. The effect of fluoxetine on fundic tone was present in the absence of the mucosa and was completely blocked by tetrodotoxin, suggesting that the source of 5-HT was neuronal. This nonvagally mediated regulation of fundic tone by endogenous 5-HT is expected to act in concert with the previously described vagally mediated cholinergic input to smooth muscle to regulate fundic tone (2, 16).

In conjunction with the effect of 5-HT on murine fundic muscle tone, 5-HT also appears to modulate neurotransmission. Electrical stimulation of murine fundic muscle strips elicited EJPs and IJPs that resulted in a contraction and relaxation, respectively. Atropine inhibits the EJP and L-NNA inhibits the IJP, suggesting that the predominant neurotransmitters involved are acetylcholine and nitric oxide, respectively (27). The data presented in this report suggest that 5-HT also participates in neurotransmission because 5-HT increased both EFS-induced EJPs and IJPs as well as the resulting contraction and relaxation. This effect of 5-HT appears to be mediated by neuronal 5-HT3 and 5-HT4 receptors. 5-HT3 receptors appear to be involved in both the generation of EJPs and IJPs because the 5-HT3 receptor antagonist MDL-72222 inhibited a significant component of the excitatory and inhibitory responses evoked by EFS. 5-HT4 receptors appear to be involved in the effect of 5-HT on IJPs because the 5-HT4 receptor antagonist GR-113808 inhibited EFS-evoked IJPs and the associated relaxation. In contrast to the 5-HT3 receptor antagonist MDL-72222, the 5-HT4 receptor antagonist GR-113808 did not alter EFS-evoked EJPs, suggesting no change in the cholinergic input to smooth muscle and that the observed increase in the EFS-evoked contractile response in the presence of GR-113808 was due to inhibition of the IJP.

In summary, 5-HT modulates murine fundic contractile activity through several different receptor subtypes. Sustained release of 5-HT maintains fundic tone through postjunctional 5-HT1B/D receptors. 5-HT3 receptors modulate excitatory neural input to murine fundic smooth muscle, and both 5-HT3 and 5-HT4 receptors modulate inhibitory neural input to murine fundic smooth muscle.


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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
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This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grants DK-52766 and DK-57061 and by a grant from Johnson & Johnson Pharmaceutical Research and Development.


   ACKNOWLEDGMENTS
 
We thank Kristy Zodrow for secretarial assistance and Simon Gibbons for helpful comments.


   FOOTNOTES
 

Address for reprint requests and other correspondence: G. Farrugia, Enteric NeuroScience Program, Div. of Gastroenterology and Hepatology, Mayo Clinic College of Medicine, 200 First St. SW, Rochester, MN 55905 (e-mail: farrugia.gianrico{at}mayo.edu)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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 REFERENCES
 

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